Abstract

INTRODUCTION

Trichophyton rubrum and Trichophyton mentagrophytescomplex, or Trichophyton spp. are the main etiologic agents of dermatophytosis in many parts of the world, including Brazil¹1. Achterman RR, White TC. A foot in the door for dermatophyte research. PLoS Pathog. 2012;8:e1002564.^,22. Di Chiacchio N, Madeira CL, Humaire CR, Silva CS, Fernandes LH, Dos Reis AL. Superficial mycoses at the Hospital do Servidor Público Municipal de São Paulo between 2005 and 2011. An Bras Dermatol. 2014;89:67-71.^,33. Godoy-Martinez P, Nunes FG, Tomimori-Yamashita J, Urrutia M, Zaror L, Silva V, et al. Onychomycosis in São Paulo, Brazil. Mycopathologia. 2009;168:111-6.. Overall, this contagious infection, commonly referred to as ringworm or tinea, is restricted to the outermost layers of the epidermis and its appendages, resulting in either a mild or intense inflammatory reaction, and in many cases, it is long lasting and difficult to treat¹1. Achterman RR, White TC. A foot in the door for dermatophyte research. PLoS Pathog. 2012;8:e1002564.. Topical antifungal agents, mainly azoles or allylamines, are currently used for the treatment of most dermatophytoses. In some cases, such as infections of the nail and hair, a systemic treatment is required⁴4. Hawkins DM, Smidt AC. Superficial fungal infections in children. Pediatr Clin North Am. 2014;61:443-55.^,55. Hay R. Superficial fungal infections. Medicine. 2009;37:610-12.^,66. Soares LA, Sardi JCO, Gullo FP, Pitangui NS, Scorzoni L, Leite FS, et al. Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products. Braz J Microbiol. 2013;44:1035-41.. However, therapeutic efficacy can be limited by antifungal side effects and/or resistance, patient non-adherence or therapy discontinuation, the cost of medication, and drug interactions⁷7. Denning DW, Hope WW. Therapy for fungal diseases: opportunities and priorities. Trends Microbiol. 2010;18:195-204.^,88. Routt ET, Jim SC, Zeichner JA, Kircik LH. What is new in fungal pharmacotherapeutics? J Drugs Dermatol. 2014;13:391-5.. Antifungal drugs have a limited number of cellular targets such as ergosterol and the enzymes involved in its synthesis, nucleic acids and the cell wall synthesis, and the formation of microtubules⁹9. Martinez-Rossi NM, Peres NT, Rossi A. Antifungal resistance mechanisms in dermatophytes. Mycopathologia. 2008;166:369-83.. Studies on compounds with potential antifungal action are important, not only for the treatment of dermatophytosis, but also to lead to the discovery of new cellular targets for the treatment of fungal infections.

Natural products have contributed significantly in healthcare since ancient times, when they have been extensively used in folk medicine for the treatment of various diseases¹⁰10. Yeung BK. Natural product drug discovery: the successful optimization of ISP-1 and halichondrin B. Curr Opin Chem Biol. 2011;15:523-8.. Indeed, the antimicrobial activity of extracts and essential oils from native plants, including their effects on dermatophytes, has been reported worldwide⁶6. Soares LA, Sardi JCO, Gullo FP, Pitangui NS, Scorzoni L, Leite FS, et al. Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products. Braz J Microbiol. 2013;44:1035-41.^,1111. Holetz FB, Pessini GL, Sanches NR, Cortez DA, Nakamura CV, Filho BP. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz. 2002; 97:1027-31.^,1212. Mabona U, Viljoen A, Shikanga E, Marston A, Van Vuuren S. Antimicrobial activity of southern African medicinal plants with dermatological relevance: from an ethnopharmacological screening approach, to combination studies and the isolation of a bioactive compound. J Ethnopharmacol. 2013;148:45-55.^,1313. Soares LA, Gullo FP, Sardi JCO, Pitangui NS, Costa-Orlandi CB, Sangalli-Leite F, et al. Anti-trichophyton activity of protocatechuates and their synergism with fluconazole. Evid Based Complement Alternat Med. 2014;2014:957860.^,1414. Souza LKH, Oliveira CMA, Ferri PH, Santos SC, Oliveira Júnior JG, Miranda ATB, et al. Antifungal properties of Brazilian cerrado plants. Braz J Microbiol. 2002;33:247-49.^,1515. Zimmermam-Franco DC, Bolutari EB, Polonini HC, do Carmo AM, Chaves M, Raposo NR. Antifungal activity of Copaifera langsdorffii Desf oleoresin against dermatophytes. Molecules. 2013;18:12561-70.. Importantly, they exhibit a large chemical diversity and biological activity, representing an alternative easily obtainable for the treatment of various diseases.

In this study, the in vitro antifungal activity of different extracts obtained from nine plant species of semiarid vegetation (caatinga) of Northeast Brazil were investigated againstT. rubrum and T. mentagrophytes complex.

MATERIAL AND METHODS

Microorganisms:T. rubrum ATCC 28189, T. mentagrophytes ATCC 11481, and 33 clinical isolates of T. rubrum (n = 24) and T. mentagrophytes complex (n = 9) were used in this study. The clinical isolates were recovered from nail and skin infections, which were obtained from the fungal collection of the Medical Mycology Laboratory of the Universidade Estadual de Maringá.

Plant material: Nine plant species of the semiarid vegetation (caatinga) of Northeast Brazil (seven native and two cultivated) were selected for this study. The voucher specimens were deposited and identified at the herbaria of the Universidade Federal de Pernambuco (UFPE), Universidade Federal do Rio Grande do Norte (UFRN), and Instituto Agronômico de Pernambuco(IPA) (Table 1).

Preparation of crude extracts: The plant parts were dried at 45^oC, until a constant weight was achieved, and ground into powder using a Willey mill (Adamo(r) ). The extract solutions were obtained by reflux using water (aqueous extract, AE), ethanol: water (1:1, v/v, ethanolic extract, EE), or acetone: water (1:1, v/v, acetone extract, AcE) as previously described¹⁶16. Ferreira MRA, Santiago RR, Langassner SMZ, de Mello JCP, Svidzinski TIE, Soares LAL. Antifungal activity of medicinal plants from Northeastern Brazil. J Med Plants Res. 2013;7:3008-13. (Table 1). Stock solutions (2,000.0 mg/mL) of crude extracts were prepared in water containing 10% dimethylsulfoxide [DMSO, v/v (Sigma-Aldrich, USA)] and twofold serial dilutions were prepared in growth medium (1,000.0 -1.95 mg/mL), and added once in each assay. The final concentration of DMSO in the assays did not exceed 1% (v/v).

In vitro antifungal assay: The minimum inhibitory concentrations (MIC) of crude extracts against dermatophytes were determined using the standard broth microdilution assay¹⁷17. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. 2nd ed. Wayne: CLSI; 2008. (CLSI M38-A2)., except for the conidia counts that were performed in a Neubauer chamber. The conidial suspensions were adjusted to obtain a final concentration ranging from 2.5 × 10³3. Godoy-Martinez P, Nunes FG, Tomimori-Yamashita J, Urrutia M, Zaror L, Silva V, et al. Onychomycosis in São Paulo, Brazil. Mycopathologia. 2009;168:111-6. to 5 × 10³3. Godoy-Martinez P, Nunes FG, Tomimori-Yamashita J, Urrutia M, Zaror L, Silva V, et al. Onychomycosis in São Paulo, Brazil. Mycopathologia. 2009;168:111-6. CFU/mL. Terbinafine was used as a quality control. Wells containing 1% DMSO (v/v) and wells without fungal cells in each plate served as growth and sterility controls, respectively. For determination of minimal fungicidal concentrations (MFC), the contents from the wells showing no growth were transferred to Sabouraud dextrose agar plates and incubated at 25 ^oC for seven days. Selected extracts (according to MIC values and quantity available for testing) were also assayed against 33 isolates of T. rubrum (n = 24) and T. mentagrophytes complex (n = 9). All experiments were performed in duplicate on two different occasions.

The antifungal activity of the extracts was ranked according to MIC values using the criteria established by SCORZONI etal.¹⁸18. Scorzoni L, Benaducci T, Almeida AMF, Silva DHS, Bolzani VS, Mendes-Giannini, MJS. Comparative study of disk diffusion and microdilution methods for evaluation of antifungal activity of natural compounds against medical yeasts Candida spp and Cryptococcus sp. Rev Cien Farm Básica Apl. 2007; 28:25-34.: MIC ≤ 75.0 µg/mL, classified as strong activity; 75.0 < MIC ≤ 150.0 µg/mL, moderate activity; 150.0 < MIC ≤ 250.0 µg/mL, weak activity, and MIC > 250.0 µg/mL, inactive.

RESULTS AND DISCUSSION

The Brazilian caatinga is one of the richest biomes in terms of biodiversity, harboring several native or cultivated plant species. Many plant species of this region are used in folk medicine or as commercial herbal to treat different conditions. However, few ethnobotanical and ethnopharmacological studies have been conducted on these medicinal plants¹⁹19. de Albuquerque UP, Muniz de Medeiros P, de Almeida AL, Monteiro JM, Machado de Freitas Lins Neto E, Gomes de Melo J, et al. Medicinal plants of the caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach. J Ethnopharmacol. 2007;114:325-54.. Moreover, antifungal activity against dermatophytes has not yet been studied for many of these plant species. Previous results by our research group have shown that the nine plant species (seven native and two cultivated) selected from this biome exhibited growth-inhibitory activity against variousCandida species¹⁶16. Ferreira MRA, Santiago RR, Langassner SMZ, de Mello JCP, Svidzinski TIE, Soares LAL. Antifungal activity of medicinal plants from Northeastern Brazil. J Med Plants Res. 2013;7:3008-13.. In this study, the antifungal activity of these plants was evaluated against T. rubrum and T. mentagrophytes complex.

First, T. rubrum ATCC 28189 and T. mentagrophytesATCC 11481 reference strains were used for the initial screening of 23 crude extracts obtained from the plant species of the Brazilian caatingaand results are reported in Table 1. The presence of 1% DMSO (v/v) did not affect fungal growth. Overall, all plants showed a fungicidal effect (MIC and MFC values of each extract were exactly at the same concentration) against either species, with MIC/MFC values of the active extracts ranging from 15.6 to 250.0 µg/mL. The highest values of MIC/MFC (1,000.0 µg/mL and 500.0 µg/mL, against both species) were observed for AE of Schinus terebinthifolius and for AE of Piptadenia colubrina,and these extracts were considered inactive according to the criteria of SCORZONIet al.¹⁸18. Scorzoni L, Benaducci T, Almeida AMF, Silva DHS, Bolzani VS, Mendes-Giannini, MJS. Comparative study of disk diffusion and microdilution methods for evaluation of antifungal activity of natural compounds against medical yeasts Candida spp and Cryptococcus sp. Rev Cien Farm Básica Apl. 2007; 28:25-34.. The lowest MIC/MFC values (15.6 µg/mL), against both species, were obtained for AcE ofParapiptadenia rigida. No differences in MIC/MFC values were observed between the solvents used for preparing the crude extracts ofLibidibia ferrea, Persea americana, Mimosa tenuiflora, and Mimosa ophthalmocentra, and except for the last species, all crude extracts displayed strong antifungal activity. For the other plants, there was a two- to 16-fold decrease in MIC/MFC values depending on the solvent used in extract preparations. Indeed, the type of solvent used during extraction can affect both the yield and the number and type of phytochemicals obtained. The aqueous mixtures of ethanol or acetone have been used to extract mainly soluble polyphenols from plant material²⁰20. Garcia-Salas P, Morales-Soto A, Segura-Carretero A, Fernández-Gutiérrez A. Phenolic-compound-extraction systems for fruit and vegetable samples. Molecules. 2010;15:8813-26.. Polyphenols comprise a large family of compounds found in several plant species, which have a chemical structure consisting of at least one phenolic ring and which display various biological properties, including antifungal activity²¹21. Negri M, Salci TP, Shinobu-Mesquita CS, Capoci IRG, Svidzinski TIE, Kioshima ES. Early state research on antifungal natural products. Molecules. 2014;19: 2925-56.. A high content of condensed tannins was detected in AcE of stem bark of P. rigida²²22. de Araújo AA, Soares LA, Ferreira MRA, de Souza Neto MA, da Silva GR, de Araújo RF Jr, et al. Quantification of polyphenols and evaluation of antimicrobial, analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of Libidibia ferrea, Parapiptadenia rigida and Psidium guajava. J Ethnopharmacol. 2014;156:88-96., which may be responsible for the antidermatophytic activity reported previously²¹21. Negri M, Salci TP, Shinobu-Mesquita CS, Capoci IRG, Svidzinski TIE, Kioshima ES. Early state research on antifungal natural products. Molecules. 2014;19: 2925-56. and in the present study. However, the presence of other phytochemical groups cannot be ruled out.

Currently, there are still few reports describing the antifungal activity of extracts or substances derived from plant species against dermatophytes⁶6. Soares LA, Sardi JCO, Gullo FP, Pitangui NS, Scorzoni L, Leite FS, et al. Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products. Braz J Microbiol. 2013;44:1035-41.^,1111. Holetz FB, Pessini GL, Sanches NR, Cortez DA, Nakamura CV, Filho BP. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz. 2002; 97:1027-31.^,1212. Mabona U, Viljoen A, Shikanga E, Marston A, Van Vuuren S. Antimicrobial activity of southern African medicinal plants with dermatological relevance: from an ethnopharmacological screening approach, to combination studies and the isolation of a bioactive compound. J Ethnopharmacol. 2013;148:45-55.^,1313. Soares LA, Gullo FP, Sardi JCO, Pitangui NS, Costa-Orlandi CB, Sangalli-Leite F, et al. Anti-trichophyton activity of protocatechuates and their synergism with fluconazole. Evid Based Complement Alternat Med. 2014;2014:957860.^,1414. Souza LKH, Oliveira CMA, Ferri PH, Santos SC, Oliveira Júnior JG, Miranda ATB, et al. Antifungal properties of Brazilian cerrado plants. Braz J Microbiol. 2002;33:247-49.^,1515. Zimmermam-Franco DC, Bolutari EB, Polonini HC, do Carmo AM, Chaves M, Raposo NR. Antifungal activity of Copaifera langsdorffii Desf oleoresin against dermatophytes. Molecules. 2013;18:12561-70.. Among the plant species analyzed here, most have shown antifungal activity againstCandida and Cryptococcus species¹¹11. Holetz FB, Pessini GL, Sanches NR, Cortez DA, Nakamura CV, Filho BP. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz. 2002; 97:1027-31.^,1616. Ferreira MRA, Santiago RR, Langassner SMZ, de Mello JCP, Svidzinski TIE, Soares LAL. Antifungal activity of medicinal plants from Northeastern Brazil. J Med Plants Res. 2013;7:3008-13.^,2323. Barbieri DS, Tonial F, Lopez PV, Sales Maia BH, Santos GD, Ribas MO, et al. Antiadherent activity of Schinus terebinthifolius and Croton urucurana extracts on in vitro biofilm formation of Candida albicans and Streptococcus mutans. Arch Oral Biol. 2014; 59:887-96.^,2424. de Souza GC, Haas AP, von Poser GL, Schapoval EE, Elisabetsky E. Ethnopharmacological studies of antimicrobial remedies in the south of Brazil. J Ethnopharmacol. 2004; 90:135-43.^,2525. Lago JH, Souza ED, Mariane B, Pascon R, Vallim MA, Martins RC, et al. Chemical and biological evaluation of essential oils from two species of Myrtaceae - Eugenia uniflora L. and Plinia trunciflora (O. Berg) Kausel. Molecules. 2011;16:9827-37.^,2626. Leite JJ, Brito EH, Cordeiro RA, Brilhante RS, Sidrim JJ, Bertini LM, et al. Chemical composition, toxicity and larvicidal and antifungal activities of Persea americana (avocado) seed extracts. Rev Soc Bras Med Trop. 2009;42:110-3.^,2727. Sampaio FC, Pereira M do S, Dias CS, Costa VC, Conde NC, Buzalaf MA. In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens. J Ethnopharmacol. 2009;124:289-94.^,2828. Schmourlo G, Mendonça-Filho RR, Alviano CS, Costa SS. Screening of antifungal agents using ethanol precipitation and bioautography of medicinal and food plants. J Ethnopharmacol. 2005; 96:563-8.. More precisely, most extracts analyzed in this study were active against four Candida species (C. albicans, C. dubliniensis, C. glabrata, and C. krusei) with a MIC range of 15.62 to 500.0 mg/mL. However, in contrast to our results, MFC values were at least four times greater than the MIC values¹⁶16. Ferreira MRA, Santiago RR, Langassner SMZ, de Mello JCP, Svidzinski TIE, Soares LAL. Antifungal activity of medicinal plants from Northeastern Brazil. J Med Plants Res. 2013;7:3008-13.. Regarding dermatophytes, only the antifungal activity of E. uniflora, L. ferrea, M. tenuiflora, and P. guajava had been previously described. An inhibitory effect on dermatophytes growth was observed for essential oils²⁹29. Lima EO, Gompertz OF, Giesbrecht AM, Paulo MQ. In vitro antifungal activity of essential oils obtained from officinal plants against dermatophytes. Mycoses. 1993;36:333-6. and EE from leaves of E. uniflora cultivated in the Brazilian cerrado¹⁴14. Souza LKH, Oliveira CMA, Ferri PH, Santos SC, Oliveira Júnior JG, Miranda ATB, et al. Antifungal properties of Brazilian cerrado plants. Braz J Microbiol. 2002;33:247-49.. In the latter study, the authors reported MIC values of EE ranging from 500.0 to 1,000.0 mg/mL for T. rubrumand T. mentagrophytes¹⁴14. Souza LKH, Oliveira CMA, Ferri PH, Santos SC, Oliveira Júnior JG, Miranda ATB, et al. Antifungal properties of Brazilian cerrado plants. Braz J Microbiol. 2002;33:247-49., which differs from our results (125.0 mg/mL for both species). Chemical analyses of E. uniflora leaves revealed the presence of terpenes, mainly oxygenated sesquiterpenes, which may be responsible for the antifungal activity²¹21. Negri M, Salci TP, Shinobu-Mesquita CS, Capoci IRG, Svidzinski TIE, Kioshima ES. Early state research on antifungal natural products. Molecules. 2014;19: 2925-56.^,2525. Lago JH, Souza ED, Mariane B, Pascon R, Vallim MA, Martins RC, et al. Chemical and biological evaluation of essential oils from two species of Myrtaceae - Eugenia uniflora L. and Plinia trunciflora (O. Berg) Kausel. Molecules. 2011;16:9827-37.^,2929. Lima EO, Gompertz OF, Giesbrecht AM, Paulo MQ. In vitro antifungal activity of essential oils obtained from officinal plants against dermatophytes. Mycoses. 1993;36:333-6.. By using an agar well-diffusion method, LIMA et al.³⁰30. Lima EO, Cury AE, Gompertz OF, Paulo MQ. Atividade antifúngica de extratos obtidos de espécies de leguminoseae contra dermatófitos. Rev Bras Cien Saúde. 1997;1:53-6. reported that 2,500.0 mg/mL of AE and EE of stem bark fromM. tenuiflora inhibited the growth of clinical isolates of four dermatophyte species (T. rubrum, T. mentagrophytes, Microscopum canis and, Epidermophyton floccosum). The antidermatophytic activity of M. tenuiflora may be attributed to tannins²¹21. Negri M, Salci TP, Shinobu-Mesquita CS, Capoci IRG, Svidzinski TIE, Kioshima ES. Early state research on antifungal natural products. Molecules. 2014;19: 2925-56., which represent the major class of compounds in stem bark of this plant³¹31. Rivera-Arce E, Gattuso M, Alvarado R, Zárate E, Agüero J, Feria I, et al. Pharmacognostical studies of the plant drug Mimosae tenuiflorae cortex. J Ethnopharmacol. 2007;113:400-8.. In the study conducted by DUTTA et al.³²32. Dutta BK, Rahman I, Das JK. In vitro study on antifungal property of common fruit plants. Biomedicine. 2000;20:187-9., tinctures from leaves and stem bark of P. guajava (which mimics the popular use) at concentrations ranging from 5 to 15% exhibited a fungicidal effect on dermatophytes. SUWANMANEE et al.³³33. Suwanmanee S, Kitisin T, Luplertlop N. In vitro screening of 10 edible Thai plants for potential antifungal properties. Evid Based Complement Alternat Med. 2014;2014:138587. reported the antidermatophytic activity of AE of leaves from P. guajavacultivated in Thailand, with MIC of 2,670.0 mg/mL, and 3,330.0 mg/mL for T. rubrum and T. mentagrophytes, respectively, which also differ from our results (125.0 mg/mL for both species). Several compounds have been detected in the leaves of P. guajava³⁴34. Gutiérrez RM, Mitchell S, Solis RV. Psidium guajava: a review of its traditional uses, phytochemistry and pharmacology. J Ethnopharmacol. 2008;117:1-27., and the antibacterial and antifungal activities may be due to the content of polyphenols, such as flavonoids, and hydrolysable and condensed tannins²¹21. Negri M, Salci TP, Shinobu-Mesquita CS, Capoci IRG, Svidzinski TIE, Kioshima ES. Early state research on antifungal natural products. Molecules. 2014;19: 2925-56.^,2222. de Araújo AA, Soares LA, Ferreira MRA, de Souza Neto MA, da Silva GR, de Araújo RF Jr, et al. Quantification of polyphenols and evaluation of antimicrobial, analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of Libidibia ferrea, Parapiptadenia rigida and Psidium guajava. J Ethnopharmacol. 2014;156:88-96.^,3535. Arima H, Danno G. Isolation of antimicrobial compounds from guava (Psidium guajava L.) and their structural elucidation. Biosci Biotechnol Biochem. 2002;66:1727-30.^,3636. Fernandes MRV, Dias ALT, Carvalho RR, Souza CRF, Oliveira WP. Antioxidant and antimicrobial activities of Psidium guajava L. spray dried extracts. Ind Crops Prod. 2014;60:39-44.^,. Condensed tannins have also been detected in the aqueous extract of L. ferrea²²22. de Araújo AA, Soares LA, Ferreira MRA, de Souza Neto MA, da Silva GR, de Araújo RF Jr, et al. Quantification of polyphenols and evaluation of antimicrobial, analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of Libidibia ferrea, Parapiptadenia rigida and Psidium guajava. J Ethnopharmacol. 2014;156:88-96., which in this study showed strong activity against both species of dermatophytes. By using the agar well-diffusion assay, LIMA et al.³⁰30. Lima EO, Cury AE, Gompertz OF, Paulo MQ. Atividade antifúngica de extratos obtidos de espécies de leguminoseae contra dermatófitos. Rev Bras Cien Saúde. 1997;1:53-6. found that 1,250.0 mg/mL AE and EE of L. ferreainhibited the growth of clinical isolates of T. rubrum, T. mentagrophytes, M. canis, and E. floccosum. Finally, SCHMOURLO et al.²⁸28. Schmourlo G, Mendonça-Filho RR, Alviano CS, Costa SS. Screening of antifungal agents using ethanol precipitation and bioautography of medicinal and food plants. J Ethnopharmacol. 2005; 96:563-8. evaluated the AE obtained by decoction of aerial parts of S. terebinthifolius on the growth of T. rubrum, by using an agar well-diffusion (1,000.0 mg/mL) and broth microdilution (10^-6 mg/mL - 1,000.0 mg/mL) methods, and the results showed no inhibitory effect for this extract. Similarly, no antidermatophytic activity was detected for the AE from the stem bark of this plant in the present study. On the other hand, the EE of bark showed strong antifungal activity with MIC/MFC of 62.5 mg/mL for both fungal species. The differences observed between the results described in the literature and the present study may be due to the conditions of plant cultivation, such as soil type and climate, which affect the production of active compounds³⁷37. Gobbo-Neto L, Lopes NP. Plantas medicinais: fatores de influência no conteúdo de metabólitos secundários. Quim Nova. 2007; 30:374-81., extraction systems for plant compounds²⁰20. Garcia-Salas P, Morales-Soto A, Segura-Carretero A, Fernández-Gutiérrez A. Phenolic-compound-extraction systems for fruit and vegetable samples. Molecules. 2010;15:8813-26., and the antifungal susceptibility testing, which can affect the MIC values of the extracts¹⁸18. Scorzoni L, Benaducci T, Almeida AMF, Silva DHS, Bolzani VS, Mendes-Giannini, MJS. Comparative study of disk diffusion and microdilution methods for evaluation of antifungal activity of natural compounds against medical yeasts Candida spp and Cryptococcus sp. Rev Cien Farm Básica Apl. 2007; 28:25-34..

According to the MIC/MFC values obtained with dermatophyte reference strains and the availability of plant extracts, E. uniflora, L. ferrea, and P. americana were also tested in clinical isolates, which were all susceptible to terbinafine (MIC ≤ 0.004 µg/mL, Table 2). Selected extracts showed a fungicidal effect (MIC=MFC) against all clinical isolates of T. rubrum and T. mentagrophytes complex, corroborating the results obtained with the reference strains. MIC/MFC values of extracts against isolates of T. rubrum ranged from 7.8 to 250.0 µg/mL for E. uniflora (AcE), 7.8 to 62.5 µg/mL for L. ferrea (AE), and 15.6 to 62.5 µg/mL for P. americana (AcE). For T. mentagrophytes complex, the MIC/MFC values of these extracts ranged from 7.8 to 62.5 µg/mL for E. uniflora, 15.6 to 62.5 µg/mL for L. ferrea, and 7.8 to 62.5 µg/mL for P. americana. Table 2 shows the MIC₅₀ and MIC₉₀ of extracts of these three plant species, for 50 and 90% growth-inhibition of all isolates, respectively. Overall, a slight increase (twofold) in the MIC₉₀ values was observed for all extracts against both fungal species. However, except for AcE of E. uniflora, which exhibited moderate activity against most clinical isolates (MIC₉₀ of 125.0 µg/mL), the AE of L. ferrea(MIC₉₀ of 62.5 µg/mL) and AcE of P. americana(MIC₉₀ of 62.5 µg/mL) showed strong activity against most isolates.

In conclusion, all the caatinga plants studied showed a fungicidal effect against T. rubrum and T. mentagrophytescomplex. Except for E. uniflora,L. ferrea, M. tenuiflora, and P. guajava, the antifungal activity of S. terebinthifolius, P. colubrina, P. rigida, M. ophthalmocentra, and P. americana against both fungal species was described for the first time. These results are useful as a preliminary step towards further antidermatophytic-guided studies of plant species from the Braziliancaatinga.

ACKNOWLEDGEMENTS

This study was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Araucária. The authors thank Dr. A. Leyva for the English editing of the manuscript.

REFERENCES

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